Double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage. They arise following exposure to ionizing radiation or radio-mimetic chemicals, and are also generated as a byproduct of normal cellular metabolism. Faithful repair of DSBs is critical for preserving genome integrity and suppressing malignant transformation. Accurate understanding of DSB repair mechanisms is not only important for understanding tumorigenesis, but also impacts tumor response to radiation therapy and most chemotherapy agents. During DSB repair, telomerase ? an enzyme normally catalyzing the extension of telomeric DNA at chromosome ends ? is capable of promiscuously adding telomeric repeats at intra-chromosomal DSBs, potentially interfering with accurate repair. Previous studies have shown that addition of telomeric repeats into intra-chromosomal regions (known as telomere sequence insertion, or TSI) causes chromosome breakage, recombination, and rearrangements. Therefore, TSI is normally suppressed to prevent genome instability. However, the role of telomerase in DSB repair has long been neglected, and it remains unknown how telomerase-mediated TSI at DSBs is suppressed in human and mammalian cells. Our recent findings strongly support that TSI is caused by erroneous addition of telomeric repeats at DSBs by telomerase. In addition, we have identified MLH1 as a TSI suppressor in telomerase-expressing cells. Using domain-specific mutations of MLH1, we also find that MLH1 recruitment to DSBs is required for suppressing TSI. The central hypothesis of this proposal is that in response to DSB induction, MLH1 localizes at intra-chromosomal break sites to prevent telomerase from adding telomeric repeats at DSBs, therefore ensuring accurate repair and protecting genome stability. The objective of this R03 proposal is to perform a second analysis of TSI and establish a defined molecular system to investigate the regulatory mechanism underlying TSI. In Aim 1, we will develop an inducible DSB repair system to study TSI. In Aim 2, we will define the roles of various DSB repair proteins in TSI suppression. Findings from the proposed research will gain novel insights and accurate understanding of DSB repair and genome instabilities, and offer guidance in developing new therapeutic strategies for tumor management.